Conference Summary
Editor's Note: At the recent EBMT 2026 Annual Meeting, Prof. Ane Altuna Mongelos from Hospital Clínic de Barcelona and IDIBAPS delivered a presentation titled "CD19/CD84 IF-BETTER dual CAR T cells demonstrate superior control of CD19-low B-ALL than single CAR19." The report explored novel pathways to overcome antigen escape in B-cell acute lymphoblastic leukemia (B-ALL) through a dual-targeting strategy. 01 Clinical Challenge: CD19 Antigen Escape Limits CAR-T Efficacy
While CAR-T cell therapy targeting CD19 has dramatically improved outcomes in B-ALL, with complete response rates (CRR) reaching 90%, antigen escape remains a major barrier to long-term efficacy. Relapse driven by the loss or downregulation of CD19 expression is a recognized mechanism of treatment failure. Although dual-targeting designs such as CD19/CD22 are currently in clinical trials, identifying additional potent partner antigens is essential for optimizing therapeutic outcomes.
02 Antigen Selection: CD84 (SLAMF5) as a Promising Partner
The research team evaluated CD84 (also known as SLAMF5) and CD71 (transferrin receptor) as potential partner antigens for CD19-based dual CAR designs.
- Expression Profile: In primary B-ALL samples, CD84 was more frequently and homogeneously expressed compared to CD71.
- Functional Validation: In vitro assays demonstrated that CD84-directed CAR-T cells showed strong dose-dependent cytotoxicity against B-ALL cell lines (Nalm6), comparable to CD19 CAR-T cells, whereas CD71-directed CAR-T cells exhibited weaker activity.
Based on these findings, CD84 was selected as the partner antigen for dual CAR-T development.
03 Structural Optimization: Resolving Auto-activation via Hinge Mutations
The team initially explored an “AND-gated” dual CAR design (Dual 1-3) requiring simultaneous recognition of both antigens. However, experiments revealed that CD84 engagement alone was sufficient to trigger cytotoxicity, leading to a loss of antigen specificity.
- Mechanistic Analysis: This was attributed to hinge domain dimerization facilitated by cysteine residues at positions 164 and 181, which form disulfide bonds.
- Solution: By mutating these cysteine residues to serine (C164S and C181S), the researchers developed the “Dual 1-mutated” construct. This modification successfully restored antigen-specific cytotoxicity, selectively killing CD19-positive B-ALL cells while sparing CD19-negative AML cells.
04 Refinement: “If-Better” Strategy for Low-Antigen Density
Maintaining the hinge mutations, the team transitioned to an “If-Better” CAR design. In this configuration, CD19 is targeted by a full second-generation CAR, while CD84 is incorporated as a co-receptor that cannot activate the T cell on its own.
- Mechanism of Action: CAR-T killing remains CD19-dependent; however, the CD84 co-receptor enhances CAR-T function specifically when CD19 expression is low.
- Construct Comparison: Two constructs were generated—Dual 4 (utilizing the 4-1BB domain) and Dual 5 (utilizing the CD28 domain). In wild-type (CD19-high) models, both constructs matched the efficacy of single CD19 CAR-T cells without negatively impacting exhaustion markers or T-cell memory subpopulation distribution.
05 Efficacy Validation: Superior Control in CD19-Low Models
To model antigen escape, a CD19-low Nalm6 cell line was generated with an approximately 12-fold reduction in CD19 expression.
- In Vitro Efficacy: At both high and low effector-to-target (E:T) ratios, Dual 4 (4-1BB) significantly outperformed single CD19 CAR-T cells in cytotoxicity assays.
- In Vivo Efficacy: In CD19-low animal models, Dual 4 achieved superior tumor control and significantly improved survival compared to single CD19 CAR-T cells, even at sub-therapeutic doses.
- Safety Profile: Cytotoxicity assays against CD34-positive hematopoietic progenitor cells showed no killing by Dual 4, indicating a favorable hematological safety profile.
Conclusion and Outlook
Prof. Ane Altuna Mongelos concluded that CD84 is a highly suitable partner antigen for B-ALL. Through precise structural engineering and the “If-Better” strategy, CD19/CD84 dual CAR-T cells effectively address the challenge of CD19 downregulation while preserving high targeting specificity. Given that CD84-directed CAR-T cells are already undergoing Phase I clinical trials for AML, this dual-targeting approach represents a promising strategy for future clinical exploration in B-ALL and B-cell lymphomas.
